Tozasertib Analogues as Inhibitors of Necroptotic ... - ACS Publications

Article Cite This: J. Med. Chem. 2018, 61, 1895−1920

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Tozasertib Analogues as Inhibitors of Necroptotic Cell Death Sam Hofmans,† Lars Devisscher,† Sofie Martens,‡,§ Dries Van Rompaey,† Kenneth Goossens,† Tatyana Divert,‡,§ Wim Nerinckx,∥,⊥ Nozomi Takahashi,‡,§ Hans De Winter,† Pieter Van Der Veken,† Vera Goossens,‡,§ Peter Vandenabeele,‡,§,# and Koen Augustyns*,† †

Laboratory of Medicinal Chemistry, University of Antwerp, Universiteitsplein 1, Wilrijk-Antwerp 2610, Belgium Molecular Signaling and Cell Death Unit, VIB Center for Inflammation Research, Technologiepark 927, Zwijnaarde-Ghent 9052, Belgium § Department of Biomedical Molecular Biology (DBMB), Ghent University, Technologiepark 927, Zwijnaarde-Ghent 9052, Belgium ∥ Unit for Medical Biotechnology, Center for Medical Biotechnology, VIB, Technologiepark 927, Zwijnaarde-Ghent 9052, Belgium ⊥ Laboratory for Protein Biochemistry and Biomolecular Engineering, Department of Biochemistry and Microbiology, Ghent University, K.L.-Ledeganckstraat 35, Ghent 9000, Belgium # Methusalem Program, Ghent University, Ghent 9000, Belgium ‡

S Supporting Information *

ABSTRACT: Receptor interacting protein kinase 1 (RIPK1) plays a crucial role in tumor necrosis factor (TNF)-induced necroptosis, suggesting that this pathway might be druggable. Most inhibitors of RIPK1 are classified as either type II or type III kinase inhibitors. This opened up some interesting perspectives for the discovery of novel inhibitors that target the active site of RIPK1. Tozasertib, a type I pan-aurora kinase (AurK) inhibitor, was found to show a very high affinity for RIPK1. Because tozasertib presents the typical structural elements of a type I kinase inhibitor, the development of structural analogues of tozasertib is a good starting point for identifying novel type I RIPK1 inhibitors. In this paper, we identified interesting inhibitors of mTNF-induced necroptosis with no significant effect on AurK A and B, resulting in no nuclear abnormalities as is the case for tozasertib. Compounds 71 and 72 outperformed tozasertib in an in vivo TNF-induced systemic inflammatory response syndrome (SIRS) mouse model.

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INTRODUCTION

Necrosis has historically been considered to be an uncontrolled form of cell death that is refractory to therapy. However, cell death research was revitalized by the understanding that necrosis can occur in a programmed and tightly regulated manner, leading to the introduction of the term necroptosis.9 Multiple forms of regulated necrosis have been characterized so far, including necroptosis, ferroptosis, oxytosis, and pyroptosis. One of the most studied forms of regulated necrosis is tumor necrosis factor receptor-1 (TNFR1) mediated necroptosis which can be induced by TNF.9 This process of TNF-mediated necroptosis is highly dependent on the function of receptor interacting protein kinase 1 (RIPK1), which was identified as a crucial kinase in the necroptotic core machinery.10−14 It should be noted that stimulation of TNFR1 by TNF does not irrevocably result in cell death. The binding of TNF to its receptor results in the formation of a receptor proximal complex I in which RIPK1 fulfills an

Cell death upon stimulation by tumor necrosis factor (TNF) mainly occurs by two major forms, namely apoptosis and necrosis. These two different forms of cell death are each characterized by their own typical morphological features.1,2 Apoptosis is the major cell death pathway used to remove unwanted and harmful cells in an immunologically “silent” way.3 The main executioners of the apoptotic pathway are the so-called caspases, a class of proteolytic enzymes that can cleave various intracellular proteins.4 Because the execution of apoptosis is highly dependent on caspase activity, it can be inhibited by using the pan-caspase inhibitor zVAD.fmk.5,6 In contrast, necroptosis is characterized by a lack of caspase activation, cytoplasmic and mitochondrial swelling, random DNA degradation, and irreversible plasma membrane damage. This in turn results in the leakage of intracellular content and the so-called “damage associated molecular patterns” (DAMPs) in the surrounding tissue which then initiate an inflammatory response.7,8 © 2018 American Chemical Society

Received: September 29, 2017 Published: February 13, 2018 1895

DOI: 10.1021/acs.jmedchem.7b01449 J. Med. Chem. 2018, 61, 1895−1920

Journal of Medicinal Chemistry

Article

Figure 1. Reported inhibitors of necroptosis: (A) type III RIPK1 inhibitors; (B) type II RIPK1 inhibitors; (C) tozasertib, a type I pan-aurora kinase inhibitor.

(Nec-1s, 2, Figure 1A), which were able to inhibit the kinase activity of RIPK1. No other molecule has been used as extensively as Nec-1s as an important tool compound in cell death research. More importantly, the discovery of Nec-1s demonstrated that the inhibition of RIPK1 could ameliorate certain conditions and diseases.20−27 Following the success of Nec-1s, significant follow-up research has been conducted which led to the identification of multiple other molecules that were subsequently classified as necrostatins.20,28−30 Despite their excellent kinase selectivity, all of the reported necrostatins struggle with multiple drawbacks such as a narrow SAR profile, moderate potency, and nonideal pharmacokinetic properties.14,18,28,31−33 These findings underline the need for the discovery of novel inhibitors of necroptosis. Cocrystallization of Nec-1s with the RIPK1 kinase domain showed that it can be classified as a type III kinase inhibitor by occupying an allosteric hydrophobic pocket created by the DLG-out conformation without interacting with any of the residues in the hinge region of RIPK1.28,34−37 Other cocrystallization studies revealed that all necrostatins bind in

important scaffolding function. Post-translational effects to this complex I can result in the downstream activation of the transcription factor NF-kB. This in turn results in the upregulation of various pro-survival genes. To maintain a prosurvival status of the cell, the kinase activity of RIPK1 is suppressed. However, when the kinase activity of RIPK1 is not suppressed, multiple cellular outcomes such as apoptosis and necroptosis become possible. This makes RIPK1 an important enzyme that functions at the crossroads between cell death and survival, having a scaffolding function that promotes cell survival while its kinase function is important for cell death signaling (both apoptosis and necroptosis).7,14−17 Because the discovery that TNF-induced necroptosis is dependent on the kinase activity of RIPK1, it became clear that this pathway might be druggable. The design of chemical inhibitors of RIPK1 could further elucidate the complex signaling cascade that drives this form of regulated necrosis. The first inhibitors of necroptosis were discovered in 2005 in a phenotypical screening to inhibit necroptotic cell death. 14,18−20 This led to the identification of both necrostatin-1 (Nec-1, 1, Figure 1A) and necrostatin-1 stable 1896



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Figure 2. Overview of tozasertib analogues. The results from the phenotypical assay of the first series of inhibitors (Supporting Information Tables S1−S3) were used to narrow down the selection of substituents for the second series of compounds. For this second series, the most preferred functional groups were used and extra variations were additionally introduced in X and Y.

tozasertib to be opportune due to its scaffold that is divergent from currently used type II and type III RIPK1 inhibitors.37 The development and screening of structural analogues of tozasertib may thus lead to new insights in the synthesis of novel RIPK1 inhibitors. A first objective in the framework of drug repurposing was thus to evaluate if tozasertib and structural analogues of tozasertib were indeed able to effectively inhibit necroptosis. Tozasertib was originally identified as a pan aurora-kinase (AurK) inhibitor. Because these kinases play an important role during mitosis and meiosis, it is obvious that these enzymes are integral for correct cell proliferation. Inhibition of AurK by tozasertib subsequently leads to cellular abnormalities which can be observed as an inhibition of cellular growth and changes in nuclear morphology and DNA content of the cells (increased nuclear area).44−46 However, in the context of necroptosis, inhibition these effects are unfavorable and should thus be preferably removed. The second objective in the framework of drug respurposing was thus to investigate whether it was possible to decrease or even completely remove these cellular and nuclear effects associated with AurK inhibition by introducing structural variations to the tozasertib scaffold. Therefore, a library of tozasertib analogues with a 2,4,6trisubstituted pyrimidine or triazine core was synthesized.

a similar manner and can therefore all be classified as type III inhibitors.28 In addition to the necrostatins, another very promising RIPK1 inhibitor was recently discovered by GlaxoSmithKline (GSK). Screening of RIPK1 against GSK’s property collection of DNA-encoded small-molecule libraries identified GSK’481 (3, Figure 1A) which was able to selectively inhibit the kinase activity of RIPK1. Upon investigation of the binding mode of GSK’481 (3), it was found that the benzylic moiety occupied the same allosteric lipophilic pocket as the necrostatins while the benzoxazepinone moiety was located near the ATP binding site. It is thus difficult to classify this compound as either a pure type II or type III kinase inhibitor.38 Lead optimization of GSK’481 resulted in the development of GSK2982772 (4, Figure 1A), which is currently in phase 2a clinical studies for psoriasis, rheumatoid arthritis, and ulcerative colitis.39 Less work has been reported considering type II RIPK1 inhibitors, however, some compounds that use this binding mode have been reported by various research groups together with some very interesting in vivo activity (5−7, Figure 1B).28,35 In an attempt to discover novel necroptosis inhibitors, we decided to use a drug repurposing approach. The investigation whether well-characterized drugs can be used in an entirely different context has received increased interest over the last years, mainly due to the rising costs of traditional drug development. Also, drugs selected for drug repurposing have often been through several stages of clinical development and therefore have a well-characterized safety profile and pharmacokinetic profile.40−42 A paper by Davis et al. published a kinase inhibitor selectivity study back in 2011, reporting a screening of 72 preclinical and clinical kinase inhibitors against 442 kinases, covering more than 80% of the human catalytic protein kinome. In this screening tozasertib (also known as VX680 or MK-0457) had the strongest affinity for RIPK1 of all tested kinase inhibitors (8, Figure 1C) with a Kd of 20 nM.43 In addition to its high affinity for RIPK1, we also considered

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RESULTS AND DISCUSSION Compound Design. Prior to the synthesis of the first library of inhibitors, the potential of tozasertib to inhibit TNFinduced necroptosis was validated. Next, a library of 30 compounds that are structurally similar to tozasertib was synthesized in order to investigate whether novel analogues of tozasertib were still able to inhibit TNF-induced necroptosis in a dose-dependent manner. On the other hand, this also helped to identify which structural modifications were preferred to possibly increase the potency of these analogues. The structural variations were introduced at three different positions. First, the amide-linked aliphatic substituent at R1 was varied between 1897



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corresponding amide analogues 59 and 60, respectively. Catalytic hydrogenation of the nitro-functional group resulted in compounds 61−62. For the sake of clarity, the second series can be divided into three smaller groups of 12 compounds, of which the structures can be described as 2,4,6-triaminosubstituted pyrimidines, 2,6diamino-4-sulfosubstituted triazines, and 2,4,6-triaminosubstituted triazines. The synthesis of these compounds is discussed in Schemes 4−6, respectively. The synthesis of 2,4,6-triaminosubstituted pyrimidines is described in Scheme 4. The pyrimidine derivates were synthesized starting from 2,4,6-trichloropyrimidine 63. First the 2-position was substituted by treating 63 with the corresponding 5-methyl-1H-pyrazol-3-amine or 4,5-dimethyl1H-pyrazol-3-amine to give intermediates 64−65. These compounds were in turn coupled to the previously prepared substituents 61−62 to give 66−69. Finally, compounds 66−69 were treated with either N-methylpiperazine, morpholine, or piperidine using the same conditions as described earlier to yield final compounds 70−81. The synthesis of 2,6-diamino-4-sulfosubstituted triazines is described in Scheme 5. All derivatives of this type were synthesized from the 2,4,6-trichlorotriazine 82. First, intermediates 13 and 14 were introduced to the triazine core, which resulted in the intermediates 83 and 84. Subsequently these intermediates were treated with either 5-methyl-1H-pyrazol-3amine or 4,5-dimethyl-1H-pyrazol-3-amine, resulting in intermediates 85−88. Once again, the N-methylpiperazine, morpholine, or piperidine moiety was introduced at the 6-position of the triazine core of compounds 85−88, which resulted in the formation of the final compounds 89−100. The synthesis of 2,4,6-triaminosubstituted triazines is described in Scheme 6. The triazine derivates were synthesized from 2,4,6-trichlorotriazine 82. In the first step, intermediates 61−62 were coupled to 82 to give compounds 101 and 102. Next, overnight treatment of 101 and 102 with the appropriate 5-methyl-1H-pyrazol-3-amine or 4,5-dimethyl-1H-pyrazol-3amine yielded compounds 103−106. Treatment of 103−106 with either N-methylpiperazine, morpholine, or piperidine using conditions as described in previous schemes afforded target triazines 107−118. Evaluation and Characterization of the Synthesized Tozasertib Analogues. Phenotypical Screening of the Compounds in L929 Cells. The research was initiated by synthesizing a preliminary library of 30 compounds in order to investigate which substituents in R1, R2, and R3 were favorable. Exact IC50 values and assay conditions are reported in Table S1 (Supporting Information, Tables S1−S3). In general, it was observed that compounds containing a cyclopropyl and cyclohexyl moiety in R1 showed the best potential toward the inhibition of TNF-induced necroptosis. Regarding R2, both hydrogen and methyl substituents were well tolerated, but generally the compounds containing a hydrogen substituent instead of an extra methyl group showed to be more potent for inhibiting TNF-induced necroptosis. At R3, it was clear that compounds which contained an N-methylpiperazine, morpholine, or piperidine moiety were most potent in inhibiting TNFinduced necroptosis (Figure 3). With these preferred groups at R1, R2, and R3 in place, additional structural variations were introduced to the central heterocyclic core and the linker connecting the two aromatic six-membered rings. A second compound library was synthesized in which nearly all possible and preferred

different cyclic and aliphatic groups with divergent steric properties. Second, the pyrazole moiety at the R2 position was preserved but its substitution pattern was varied. Finally, the solubility-improving moiety at R3 was varied with different cyclic amines (Figure 2). These 30 compounds were tested in a phenotypical assay in which their inhibitory potential against TNF-induced necroptosis was studied (Supporting Information, Tables S1−S3). The results from this initial series allowed us to formulate a preliminary structure−activity relationship (SAR) to see which substituents in R1, R2, and R3 were beneficial for improving the inhibitory potency of this type of compounds. This newly found SAR was then used as a guideline in the design of a second library of tozasertib analogues. Next to these most preferred functional groups in R1, R2, and R3, additional variations were introduced to the central heterocyclic core and linker atom connecting the two six-membered aromatic systems (Figure 2, respectively shown as X and Y). The introduction of these modifications provided a set of 46 final compounds, 8, 43, 44, 46, 48, 50, 52, 53, 55, 57, 70−81, 89−100, and 107−118. These compounds were evaluated for their potential to inhibit mTNF-induced necroptosis in the presence of zVAD.fmk. Because tozasertib analogues can still potentially interfere with cell proliferation and induce nuclear abnormalities, the effect of these 46 compounds on cellular growth and nuclear area was also investigated. Chemistry. All compounds in this study were prepared following the general strategies in Schemes 1−6. In general, nearly all final compounds were prepared using nucleophilic aromatic substitution reactions. Prior to the synthesis of the first library of 30 tozasertib analogues, several N-(4-mercaptophenyl)amide intermediates 10−14 were synthesized (Scheme 1). These molecules were Scheme 1a

a

Reagents and conditions: (a) 4-aminobenzothiol, substituted acylchloride, triethylamine, tetrahydrofuran, 2 h, 0 °C.47

synthesized from the commercially available 4-aminobenzothiol 9 which was acylated using appropriate acyl chlorides to form the corresponding aromatic amides. Treatment of 4,6-dichloro-2-(methylsulfonyl)pyrimidine 15 with intermediates 10−14 resulted in compounds 16−20 (Scheme 2). These compounds were subsequently treated with 5-methyl-1H-pyrazol-3-amine or 4,5-dimethyl-1H-pyrazol-3amine, providing compounds 21−27. Target compounds 8 and 28−57 were obtained by substituting the 6-position of the pyrimidine scaffold with a a piperidine, morpholine, piperazine, N-methyl- or N-ethylpiperazine. To investigate the effects of the heteroatom that links both aromatic six-membered rings in tozasertib, we prepared a set of various N-(4-aminophenyl)cycloalkyl carboxamide intermediates which are described in Scheme 3. These compounds were prepared by N-acylation of 58 using either cyclopropanecarbonyl chloride or cyclohexanecarbonyl chloride to afford the 1898



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Scheme 2a

a Reagents and conditions: (a) 4,6-dichloro-2-(methylsulfonyl)pyrimidine, triethylamine, acetonitrile, 2 h, −10 °C to rt; (b) 5-methyl-1H-pyrazol-3amine or 4,5-dimethyl-1H-pyrazol-3-amine, DIPEA, DMF, 18 h, 90 °C; (c) piperidine, morpholine, piperazine, N-methylpiperazine or Nethylpiperazine, 2 h, 110 °C.47−49

Scheme 3a

a

Reagents and conditions: (a) substituted acylchloride, DIPEA, DCM, 3 h, rt; (b) Pd(OH)2, H2, methanol, 17 h, rt.50

Scheme 4a

a

Reagents and conditions: (a) 5-methyl-1H-pyrazol-3-amine or 4,5-dimethyl-1H-pyrazol-3-amine, dioxane, 12 h, rt;51 (b) 61,62, p-toluene sulfonic acid monohydrate, n-butanol, 12 h, reflux;52 (c) piperidine, morpholine, N-methylpiperazine, 2 h, 110 °C.49

aforementioned combinations were introduced. This library consists of 46 compounds, of which 8, 43, 44, 46, 48, 50, 52,

53, 55, and 57 were reused from the initial series and of which 70−81, 89−100, and 107−118 were synthesized additionally. 1899



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Scheme 5a

Reagents and conditions: (a) compound 13 or 14, DIPEA, 0 °C, dropwise addition; (b) 5-methyl-1H-pyrazol-3-amine, DIPEA, dioxane, 16 h, 120 °C; (c) 4,5-dimethyl-1H-pyrazol-3-amine, DIPEA, dioxane, 16 h, rt; (d) piperidine, morpholine, or N-methylpiperazine, 30 min, rt.

a

Scheme 6a

a Reagents and conditions: (a) 61, 62, K2CO3, dioxane, rt; (b) 5-methyl-1H-pyrazol-3-amine or 4,5-dimethyl-1H-pyrazol-3-amine, DIPEA, dioxane, 16 h, 120° C; (c) piperidine, morpholine, N-methylpiperazine, 2 h, 110°C.49

compounds is reported in the Supporting Information (Tables S2, S5−S9). Because tozasertib was initially identified as a pan-AurK inhibitor to treat multiple types of leukemia, it is to be expected that novel analogues that are derived from tozasertib can still display similar properties.48,49 As can be viewed in Table 1, tozasertib clearly reduced cellular growth (IC50,growth = 0.97 μM) and also interfered with normal cell division which can be verified by morphological analysis of the nuclei of the cells (IC50,nuclear area = 1.06 μM). Tozasertib is able to inhibit necroptosis in the low micromolar range (IC50,−0.5h = 0.98 μM and IC50,−24h = 1.02 μM). However, due to its effects on

These 46 compounds were evaluated in a phenotypical assay for their potential to inhibit necroptosis stimulated by mTNF. Additional data on different cell lines and the mechanism of action of selected compounds will be published elsewhere.53 As mentioned earlier, treatment of cells with tozasertib results in an inhibition of TNF-induced necroptosis but also affects cellular growth and induces nuclear abnormalities with similar IC50 values. With this second library, we investigated whether it was possible for a molecule to be a potent inhibitor of TNF-induced necroptosis without affecting the cellular growth and/or the nuclear area. The results of this assay are reported in Table 1. The 95% confidence interval for the active 1900



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Figure 3. Summary of the results from the first phenotypical assay in determining the preferred substituents in R1, R2, and R3.

induced necroptosis. In this enzymatic assay, some interesting observations were seen regarding the nature of the linker atom between the two six-membered aromatic rings. To investigate this further, the structural effects of the substitution of a sulfur to a nitrogen linker were studied through a molecular modeling study. Evaluation of the Compounds against the Recombinant Enzymes. In addition to compounds 70−72 and 97, Nec-1s (2) and tozasertib (8) were also included as reference compounds (Table 2). In this enzymatic assay, Nec-1s (2) potently inhibited hRIPK1 but did not affect the hAurK enzymes. Tozasertib (8) on the other hand was shown to be a potent inhibitor of all the enzymes included in this assay. In this experiment, compounds 70−72 were able to inhibit hRIPK1 with low micromolar IC50 values. These compounds are even 2−4-fold more potent for inhibition of hRIPK1 than Nec-1s (2). Tozasertib (8) was also shown to be a very potent inhibitor of both hAurK A and hAurK B. It should be noted that the most potent compounds 70−72 still inhibited both hAurK enzymes, nevertheless, in a significantly less potent manner than tozasertib. Compound 97, which did not demonstrate any potency toward the inhibition of TNFinduced necroptosis in the aforementioned phenotypic assay, showed to be a significantly less potent inhibitor for hRIPK1 in the enzymatic assay. Compound 97 still maintained its inhibition of the hAurK enzymes but in a slightly more potent manner in comparison to compound 70−72. In general, it can be concluded that a correlation is observed between the results of both the phenotypic and the enzymatic assay. Possible Effects of the Nature of the Linking Atom X. Inspection of the tozasertib:aurora kinase A:TPX2 cocrystal structure (PDB 3E5A) reveals that the linker atom in tozasertib (8) is rather solvent-exposed and does not engage in highly specific interactions with the protein.54 It thus seems unlikely that contacts with the protein are responsible for the difference in assay outcomes. However, the linker atoms do differ substantially in electronic properties. The nitrogen linker is more strongly conjugated to the aromatic ring systems, which may increase the rigidity of the general structure and influence the conformational preferences of the system. To investigate this effect, two model systems were designed: N-phenylpyrimidin-2-amine and 2-(phenylthio)pyrimidine. The two torsion angles around the nitrogen or sulfur linker atoms controlling the orientation of the two rings were examined through relaxed dihedral scans at the PW6B95-D3(BJ)55−57/ def2-TVZP58,59 level of theory in ORCA.60 For the first

cell division, tozasertib can be classified as an aselective compound for the inhibition of necroptosis. To meet the aforementioned goals, it was important that tozasertib analogues were free from these undesirable effects on cell growth and nuclear area while still maintaining a potent inhibition of TNF-induced necroptosis. Out of the 46 compounds that were tested under these conditions, seven compounds (70, 71, 72, 77, 78, 90, and 108) had the potential to inhibit TNF-induced necroptosis without affecting cell growth and/or nuclear area. Out of these seven compounds, 70−72 were able to inhibit necroptosis in a slightly more potent manner than tozasertib after both 0.5 h and 24 h pretreatment. Compounds 77, 78, 90, and 108 also provided the desired inhibitory profile albeit in a slightly less significant manner than compounds 70−72. In addition to compounds that favor necroptosis inhibition without affecting cell growth and/or nuclear area, there were also compounds with the opposite effect. These compounds did not inhibit TNF-induced necroptosis but did inhibit cellular growth and nuclear area in a very similar manner as tozasertib. This is most clearly exemplified by compound 97, which affects cell growth and nuclear area in a more potent fashion than tozasertib. The results of this assay led to another interesting remark that can be made toward the nature of the linker atom X between the two aromatic six-membered rings. In compounds 70−72, which were deemed the most interesting compounds regarding both potency and absence of nuclear abnormalities, the sulfur linker connecting the two six-membered aromatic systems was replaced by a nitrogen linker. In general, most compounds in which the sulfur linker was replaced by a nitrogen no longer affect cellular growth and nuclear area. This strongly suggests that the choice of the linker atom could be important for diminishing the unwanted cellular effects that are classically associated with AurK inhibition by tozasertib. Overall, a trend was observed that compounds containing a central triazine core, a 4,5-dimethylated pyrazole moiety at R2, or the combination of both, no longer affected cell growth and nuclear area. This was clearly exemplified by compounds 73− 75, 79−81, 92−94, and 110−112. It should be noted however that these compounds do no longer possess any activity in the phenotypical assays. To validate the findings of the phenotypical assay, the most favorable necroptosis inhibitors 70−72 were first evaluated in an enzymatic assay against recombinant hAurK A and B and hRIPK1. Compound 97 was also included in this assay because it showed a strong potential for induction of nuclear abnormalities without showing any potency to inhibit TNF1901



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Table 1. Evaluation of the Antinecroptotic Activity and Observation of Possible Effects on Cellular Growth and Nuclear Area of the Synthesized Tozasertib Analoguesa

IC50 values (μM) compd

core Y

linker X

R1

R2

R3

−0.5 h

8, tozasertib (reference) 43 44 47 48 50 52 53 55 57 70 71 72 73 74 75 76 77 78 79 80 81 89 90 91 92 93 94 95 96 97 98 99 100 107 108 109 110 111 112 113 114 115 116 117 118

CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH N N N N N N N N N N N N N N N N N N N N N N N N

S S S S S S S S S S NH NH NH NH NH NH NH NH NH NH NH NH S S S S S S S S S S S S NH NH NH NH NH NH NH NH NH NH NH NH

cyclopropyl cyclopropyl cyclopropyl cyclohexyl cyclohexyl cyclohexyl cyclohexyl cyclohexyl cyclohexyl cyclopropyl cyclopropyl cyclopropyl cyclopropyl cyclopropyl cyclopropyl cyclopropyl cyclohexyl cyclohexyl cyclohexyl cyclohexyl cyclohexyl cyclohexyl cyclopropyl cyclopropyl cyclopropyl cyclopropyl cyclopropyl cyclopropyl cyclohexyl cyclohexyl cyclohexyl cyclohexyl cyclohexyl cyclohexyl cyclopropyl cyclopropyl cyclopropyl cyclopropyl cyclopropyl cyclopropyl cyclohexyl cyclohexyl cyclohexyl cyclohexyl cyclohexyl cyclohexyl

H H H H H H Me Me Me Me H H H Me Me Me H H H Me Me Me H H H Me Me Me H H H Me Me Me H H H Me Me Me H H H Me Me Me

NMe CH2 O CH2 O NMe CH2 O NMe NEt CH2 O NMe CH2 O NMe CH2 O NMe CH2 O NMe CH2 O NMe CH2 O NMe CH2 O NMe CH2 O NMe CH2 O NMe CH2 O NMe CH2 O NMe CH2 O NMe

0.98 0.75 0.39 1.75 0.38 >3 >3 >3 >3 >3 0.53 0.62 1.04 >3 >3 >3 >3 0.64 0.91 >3 >3 >3 >3 0.70 >3 >3 >3 >3 >3 >3 >3 >3 >3 >3 >3 1.06 >3 >3 >3 >3 >3 >3 >3 >3 >3 >3

b

−24 h 1.02 1.74 0.81 5.04 0.36 >3 >3 >3 >3 >3 0.68 0.43 0.64 >3 >3 >3 1.94 1.50 1.75 >3 >3 >3 >3 1.45 1.14 >3 >3 >3 >3 1.63 >3 >3 >3 >3 >3 2.64 >3 >3 >3 >3 >3 >3 >3 >3 >3 >3

c

cell growthd

nuclear areae

0.97 1.32 1.00 0.813 0.214 3 >3 >3 >3 >3 >3 >3 >3 >3 >3 >3 2.25 >3 >3 >3 >3 >3 >3 0.44 >3 >3 >3 >3 2.17 0.33 >3 >3 >3 >3 >3 >3 >3 >3 >3 >3 2.30 >3 >3 >3 >3

1.06 1.00 1.04 >3 3 >3 >3 >3 >3 >3 >3 >3 >3 >3 >3 >3 >3 >3 >3 >3 >3 >3 >3 0.80 >3 >3 >3 >3 >3 0.40 >3 >3 >3 >3 >3 >3 >3 >3 >3 >3 >3 >3 >3 >3 >3

Compound is classified as inactive when less than 50% inhibition is observed at a concentration of 3 μM of inhibitor. The IC50 value is then shown as >3 μM in Table 4.1. bInhibition of mTNF-induced necroptosis in the presence of pan-caspase inhibitor zVAD.fmk. L929sAhFAS cells have been pretreated with compound for 0.5 h followed by stimulation with mTNF (2500 IU/mL) and zVAD.fmk (1 μM) for 3 h in the presence of Hoechst (1 μM) and propidium iodide (PI) (3 μM). IC50 values were calculated through a dose−response curve with the percentage of cell death as

a

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Table 1. continued quantitative read-out. cInhibition of mTNF-induced necroptosis in the presence of pan-caspase inhibitor zVAD.fmk. L929sAhFAS cells have been pretreated with compound for 24 h followed by stimulation with mTNF (2500 IU/mL) and zVAD.fmk p(1 μM) for 3 h in the presence of Hoechst (1 μM) and propidium iodide (PI) (3 μM). IC50 values were calculated through a dose−response curve with the percentage of cell death as quantitative read-out. dValue is calculated via dose−response curve of the concentration of the individual compound in function of the cellular growth after 24 h. For curve fitting and IC50 determination, the bottom value equals the total amount of cells in the highest concentration of tozasertib and the top value equals the total amount of cells in untreated conditions. eValue is calculated via dose−response curve of the concentration of the individual compound in function of the mean nuclear area. For curve fitting and IC50 determination, this results in a sigmoidal curve of which the bottom value equals the normal nuclear area and the top value equals the maximal nuclear area at the highest concentration of tozasertib.

Table 2. IC50 Values from the Enzymatic Assaya compd

hAurK A (μM)

hAurK B (μM)

hRIPK1 (μM)

Nec-1s (2) tozasertib (8) 70 71 72 97

>3 0.030 0.310 0.430 0.293 0.210

>3 0.068 0.959 0.853 1.345 0.229

0.754 0.208 0.295 0.167 0.178 2.468

dihedral is approximately 336°. As this is quite close to a minimum, we do not anticipate this dihedral to be responsible for the differences observed in the cellular assay. For the second dihedral, illustrated in Figure 4 (bottom), marked differences between the energy profiles are observed. For the sulfur-linked system, mimima are located at 90° and 270°. The angle in the crystal structure is 98°, corresponding to a minimum. In the most stable conformation of the sulfur-linked system, the two aromatic rings are approximately peripendicular to one another. In the nitrogen-linked system, energetic minima are located at 0° (and 360°) and 180° degrees. The 98° torsion angle in the crystal structure of tozasertib (8) is highly unfavorable for this system. In contrast to the sulfur-linked system, the most stable conformation of the nitrogen-linked system is a fully planar conformation. We thus propose that the nitrogen-linked compounds are not able to bind in the same conformation as tozasertib (8) due to the difference in their respective

a

Compounds were evaluated for their potency to inhibit recombinant hAurK A, hAurK B, and hRIPK1. The results were obtained through a luminescence-based ADP-Glo kinase assay. When a compound was inactive, the IC50 value is shown as >3 μM. The confidence intervals and statistical validation of the results presented in Table 2 are reported in the Supporting Information, Table S3.53

dihedral, depicted in Figure 4 (top), both profiles are similar in shape. Mimima are located at 0° (or 360°) and 180°. In the tozasertib:aurora kinase A:TPX2 cocrystal structure, this

Figure 4. DFT relaxed surface scan of both dihedrals. (top) Relaxed surface scan for the first dihedral. (bottom) Relaxed surface scan for the second dihedral. The energies for the nitrogen-linked model compound are shown in blue, and the energies for the sulfur-linked model compound are shown in orange. The arrow denotes the dihedral angle being scanned. Dihedral angle values corresponding to the crystal structure are marked by a dotted line. 1903



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conformational preferences. This may explain the lower affinity of the nitrogen-linked analogues for AurK A. Investigation of the Binding Mode of Tozasertib in RIPK1. A docking experiment was performed to propose a possible binding mode for tozasertib in RIPK1 (Figure 5).

Figure 6. Tozasertib and its analogues 71 and 72 protect against TNFinduced SIRS. Mice were challenged with 10 μg of mTNF (500 μg/ kg) in the presence or absence of tozasertib/analogue. All compounds were given by oral gavage 1.5 h before mTNF challenge at a dose of 50 mg/kg. A Mantel−Cox test was performed as statistical analysis on the survival curves. P-values

Tozasertib Analogues as Inhibitors of Necroptotic ... - ACS Publications

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